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. 2010 Dec 15;99(12):4037-46.
doi: 10.1016/j.bpj.2010.10.025.

Multiplexing of multicolor bioluminescence resonance energy transfer

Billy Breton  1 , Étienne Sauvageau, Joris Zhou, Hélène Bonin, Christian Le Gouill, Michel Bouvier
Affiliations

Multiplexing of multicolor bioluminescence resonance energy transfer

Billy Breton et al. Biophys J. .

Abstract

Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.

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Figures

Figure 1
Figure 1
Bioluminescence and fluorescence emission spectra. Bioluminescence emission spectra of cells overexpressing EBFP2-EPAC-Rluc2 (A, blue line), SCFP3A-EPAC-Rluc2 (B, cyan line), GFP10-EPAC-Rluc2 (C, green line), and mAmetrine-EPAC-Rluc2 (D, dark yellow line), and negative control mCherry-EPAC-Rluc2 (A–D, black dotted line) were measured after the addition of coel-400a. (E) The bioluminescence of the mCherry-EPAC-Rluc2 control (which was not different from the emission from Rluc2 alone, since mCherry is not an acceptor for the Rluc2/coel-400a) was subtracted from the emission spectrum for each FP-EPAC-Rluc2 fusion proteins and normalized as a ratio of the maximal emission for each FP. (F) Fluorescence spectra obtained from cells overexpressing each of the FP-EPAC-Rluc2 fusion proteins was measured after direct excitation at 400 nm. The curves were generated using the LOWESS fitting equation from the Prism 4.0 software.
Figure 2
Figure 2
Multicolor BRET measurements. (A–D) BRET between V2R-Rluc2 and GFP10-Gγ2 (green), EBFP2-Gγ2 (blue), SCFP3A-Gγ2 (cyan), or mAmetrine-Gγ2 (dark yellow) were measured in cells coexpressing the indicated BRET partners and Gα12, in the presence or absence of increasing concentrations of AVP. Cells were stimulated for 20 min with the indicated concentration of AVP, and coel-400a was added 10 min before the readings were taken. BRET was measured using four different filter sets (see characteristics in the Materials and Methods section), as indicated in the panels. Results are expressed as the means ± SE of three independent experiments performed in triplicate. Curves were analyzed using a nonlinear regression sigmoid fit from the Prism 4.0 software. The EC50 (nM) is indicated for each FP within each filter set. (E) The maximal AVP-promoted BRET for each pair is plotted as a function of their basal BRET signals. The linear correlations for the four filter sets were obtained using a linear regression fit from the Prism 4.0 software and are depicted by the four lines in the graph.
Figure 3
Figure 3
Multiplexing BRET. BRET400-BFP (A) and BRET400-mAmetrine (B) were measured in cells expressing the indicated combination of V2R-Rluc2, EBFP2-Gγ2, and mAmetrine-EPAC-Rluc2. BRET was measured in the absence of ligand (Ctl) or after 20 min stimulation with AVP (100 nM) or forskolin (100 μM). Coel-400a was added 10 min before readings were taken, using a single energy donor filter (410 ± 70 nm) and two different energy acceptor filters (480 ± 20 nm for BRET400-BFP and 550LP for BRET400-mAmetrine). Two-way ANOVAs followed by Bonferroni post-tests were used to assess the statistical significance of the differences (∗∗p < 0.001) using Prism 4.0 software. (C) BRET400-BFP between V2R-Rluc2 and EBFP2-Gγ2 (squares) and BRET400-mAmetrine for the cAMP biosensor mAmetrine-EPAC-Rluc2 (circles) were measured simultaneously in cells coexpressing these constructs, after 20 min stimulation with increasing concentration of AVP. The results are expressed as the means ± SE of three independent experiments performed in triplicate. The curves were generated using the nonlinear regression sigmoid fit from Prism 4.0 software.
Figure 4
Figure 4
Multiplexing Gαi or Gαq activation with cAMP production. BRET from cells expressing (A) D2 dopamine receptor, Gαi1-91Rluc2, mAmetrine-Gγ2, and EBFP2-EPAC-Rluc2; or (B) TPαR, Gαq-121Rluc2, mAmetrine-Gγ1, and EBFP2-EPAC-Rluc2 was measured either in the absence of ligand or after 20 min stimulation with quimpirol (quim, 100 nM, A), U46619 (100 nM, B), and/or forskolin (Fsk, 100 μM, A and B), as indicated. For the negative control in A, PTX (100 ng/mL) was added 16 h before the experiment. Coel-400a was added 10 min before readings were taken, using a single energy donor filter (410 ± 70 nm) and two different energy acceptor filters (480 ± 20 nm for BRET400-BFP and 550LP for BRET400-mAmetrine). Two-way ANOVAs followed by Bonferroni post-tests were used to assess the statistical significance of the differences (∗p < 0.01, ∗∗p < 0.001) using Prism 4.0 software.
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